The long-term goal of this project is to understand the cellular mechanisms that influence the release of calcium from the sarcoplasmic reticulum (s.r.) of skeletal muscle. As the level of force production in muscle depends on the concentration of cytoplasmic calcium reached process is essential to understanding how muscle cells are activated both normally and in diseased states. The work will investigate the role of protein phosphorylation in regulating calcium release channel activity. The specific aims are 1) to investigate the influence of phosphorylation on the activity of single s.r. calcium release channels and 2) to determine whether phosphorylation plays a physiologically important role in regulating calcium release from single muscle cells. Additional experiments are designed to describe the conductance selectivity and gating properties of the s.r. calcium channel under physiological conditions. The project will use two basic experimental approaches. Single channel patch clamp recording will be used to study the behavior of the calcium release channel from frog skeletal muscle fibers. The s.r. membrane will be exposed by removing the surface membrane of isolated single fibers and then causing the fiber to contract to very short lengths. Under these conditions s.r. membrane is extruded from the surface of the fiber and can be approached with patch microelectrodes. The response of a more intact preparation will also be studied using tension generation or calcium indicator dyes to monitor calcium release from single cells ("skinned fibers").